Abstract
Covalent modifications of proteins by ubiquitin and the Small Ubiquitin-like MOdifier (SUMO) have been revealed to be involved in a plethora of cellular processes, including transcription, DNA repair and DNA damage responses. It has been well known that in response to DNA damage that blocks transcription elongation, Rpb1, the largest subunit of RNA polymerase II (Pol II), is ubiquitylated and subsequently degraded in mammalian and yeast cells. However, it is still an enigma regarding how Pol II responds to damaged DNA and conveys signal(s) for DNA damage-related cellular processes. We found that Rpb1 is also sumoylated in yeast cells upon UV radiation or impairment of transcription elongation, and this modification is independent of DNA damage checkpoint activation. Ubc9, an E2 SUMO conjugase, and Siz1, an E3 SUMO ligase, play important roles in Rpb1 sumoylation. K1487, which is located in the acidic linker region between the C-terminal domain and the globular domain of Rpb1, is the major sumoylation site. Rpb1 sumoylation is not affected by its ubiquitylation, and vice versa, indicating that the two processes do not crosstalk. Abolishment of Rpb1 sumoylation at K1487 does not affect transcription elongation or transcription coupled repair (TCR) of UV-induced DNA damage. However, deficiency in TCR enhances UV-induced Rpb1 sumoylation, presumably due to the persistence of transcription-blocking DNA lesions in the transcribed strand of a gene. Remarkably, abolishment of Rpb1 sumoylation at K1487 causes enhanced and prolonged UV-induced phosphorylation of Rad53, especially in TCR-deficient cells, suggesting that the sumoylation plays a role in restraining the DNA damage checkpoint response caused by transcription-blocking lesions. Our results demonstrate a novel covalent modification of Rpb1 in response to UV induced DNA damage or transcriptional impairment, and unravel an important link between the modification and the DNA damage checkpoint response.
Highlights
The integrity of cellular DNA is constantly challenged by both endogenous and exogenous sources, including oxygen radicals within cells, environmental UV light, ionizing radiation and other genotoxic agents [1]
When the immunoprecipitated Rpb1 was probed with an antiSUMO antibody, several bands could be seen in the UV irradiated samples, but not in the unirradiated ones (Fig. 1 A), indicating that Rpb1 was sumoylated in response to UV-induced DNA damage
Several bands could be detected in the UV irradiated sample, but not in the unirradiated one (Fig. 1B), indicating that Rpb1 is sumoylated in response to UV-induced DNA damage
Summary
The integrity of cellular DNA is constantly challenged by both endogenous and exogenous sources, including oxygen radicals within cells, environmental UV light, ionizing radiation and other genotoxic agents [1]. A major component of the response is the DNA damage checkpoint, which arrests the cell cycle to provide time for carrying out DNA repair. Mec, the counterpart of mammalian ATR (Ataxia-Telangiectasia mutated and Rad3-related), and Tel, the counterpart of mammalian ATM (Ataxia-Telangiectasia Mutated), are the kinases that sense DNA damage [2]. Mec is activated by long 39-ended single stranded DNA (ssDNA) tails generated during resection of double strand breaks or by ssDNA gaps arising in repair. The counterpart of the mammalian Chk, is the major effector of the DNA damage checkpoint, and its phosphorylation by Mec has been considered a hallmark of checkpoint activation in yeast. Phosphorylated Rad targets a number of substrate proteins, resulting in stabilization of stalled replisomes, suppression of recombination, and prevention of cell cycle progression [2]
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